Loading of the human 9-1-1 checkpoint complex onto DNA by the checkpoint clamp loader hRad17-replication factor C complex in vitro
Loading of the human 9-1-1 checkpoint complex onto DNA by the checkpoint clamp loader hRad17-replication factor C complex in vitro
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DOI：
10.1073/pnas.0437927100
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发表时间：
2003-02-18
影响因子：
11.1
通讯作者：
Sancar, A
中科院分区：
文献类型：
--
作者：
Bermudez, VP;Lindsey-Boltz, LA;Sancar, A
人类DNA损伤传感器，Rad 17复制因子C(Rad17-RFC)和Rad9-Rad1-Hus1(9-1-1)检查点复合体，被认为参与了DNA损伤检查点反应的早期步骤。Rad17-RFC和9-1-1复合体在结构上分别与复制因子、RFC钳夹和增殖细胞核抗原聚合酶钳相似。在这里，我们证明了复制和检查点钳制加载器/DNA钳制对之间的功能相似性。当两个检查点复合体的全部8个亚基在昆虫细胞中共表达时，在体内形成一个稳定的Rad17-RFC/9-1-1检查点超复合体，并且易于纯化。这两个单独纯化的检查点复合体在体外也形成了一个超复合体，它依赖于ATP，并通过Rad17和Rad9之间的相互作用来调节。Rad17-RFC与缺口环状、缝隙和启动的DNA结合，并以ATP依赖的方式招募9-1-1复合体。反应产物的电子显微镜分析表明，9-1-1环被夹在DNA周围。
The human DNA damage sensors, Rad 17-replication factor C (Rad17-RFC) and the Rad9-Rad1-Hus1 (9-1-1) checkpoint complex, are thought to be involved in the early steps of the DNA damage checkpoint response. Rad17-RFC and the 9-1-1 complex have been shown to be structurally similar to the replication factors, RFC clamp loader and proliferating cell nuclear antigen polymerase clamp, respectively. Here, we demonstrate functional similarities between the replication and checkpoint clamp loader/DNA clamp pairs. When all eight subunits of the two checkpoint complexes are coexpressed in insect cells, a stable Rad17-RFC/9-1-1 checkpoint supercomplex forms in vivo and is readily purified. The two individually purified checkpoint complexes also form a supercomplex in vitro, which depends on ATP and is mediated by interactions between Rad17 and Rad9. Rad17-RFC binds to nicked circular, gapped, and primed DNA and recruits the 9-1-1 complex in an ATP-dependent manner. Electron microscopic analyses of the reaction products indicate that the 9-1-1 ring is clamped around the DNA.